2. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is selected from unsubstituted (C1-C18) alkyl,
alkenyl and alkynyl.

3. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted aryl.

4. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18) oxyl.

5. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18)
alkylcarboxyl or arylcarboxyl.

6. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18) ester.

7. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18) carbonyl.

8. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted alkyl(C1-C18) or aryl
amino.

9. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18) alkyl or
aryl nitro.

10. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18) nitroso.

11. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted oxime.

12. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18) hydrazone.

13. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted azo.

14. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18) thiol.

15. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18) sulfonic
acid.

16. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is halide.

17. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted hydroxylamine.

18. A composition according to claim 1, wherein at least one of R1,
R2 and R3 is substituted or unsubstituted (C1-C18)
phosphoester.

19. A method for reducing oxidative damage to, or delaying senescence of a
cell comprising the step of contacting a cell subject to or at risk of
undesirable oxidative damage or senescence with a composition according
to claim 1.

20. A method for reducing oxidative damage to, of delaying senescence of a
cell comprising the steps of:identifying a cell as subject to or at risk
of undesirable oxidative damage or senescence; andcontacting the cell
with a composition according to claim 1.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001]This application is a continuation of and claims priority under 35
U.S.C. § 120 to U.S. Ser. No. 10/713,432, filed Nov. 13, 2003, which
is a continuation of and claims priority under 35 U.S.C. § 120 to
U.S. Ser. No. 10/038,135, filed Oct. 20, 2001, which is a continuation of
and claims priority under 35 U.S.C. § 120 to U.S. Ser. No.
09/429,412, filed Oct. 28, 1999, now U.S. Pat. No. 6,455,589, which are
incorporated herein by reference.

INTRODUCTION

[0003]1. Field of the Invention

[0004]The field of the invention is pharmaceutical compositions of primary
N-hydroxylamines.

[0005]2. Background of the Invention

[0006]-Phenyl-N-t-butyl nitrone (PBN) is one of the most widely used spin
trapping agents for investigating the existence of free radicals in
biological systems. PBN reverses the age-related oxidative changes in the
brains of old gerbils (1,2) and delays senescence in
senescence-accelerated mice (3) and in normal mice (4). PBN also delays
senescence in the normal human lung fibroblast cell line IMR90 (5). In
addition, PBN reverses mitochondrial decay in the liver of old rats (38)
and exerts a neuroprotective effect in gerbils (1,7) and rats (8,9) after
oxidative damage from ischemia/reperfusion injury. The mechanism
underlying the biological activity of PBN is still controversial.
However, PBN is a well known scavenger of radical species, though a
variety of other well known spin trap or anti-oxidants do not mimic its
anti-senescence activity in IMR90. PBN at relatively high concentrations
reduces the production of hydrogen peroxide in mitochondrial preparations
of cerebral cortex (10) and therefore may exert similar properties in
vivo. This suggests that PBN possesses special properties that do not
exist in other spin traps or antioxidants.

[0007]In the course of our study of the affect of PBN on IMR90 cells we
observed that old solutions were more effective than fresh solutions in
delaying senescence of IMR90 cells. This raised the question about the
interaction of PBN's decomposition products with IMR90 cells. This
encouraged us to test the anti-senescent effect of the PBN decomposition
products, N-t-butyl hydroxylamine and benzaldehyde on IMR90 cells. PBN
(or PBN/-OH) has been reported to decompose to N-t-butyl
hydroxylamine or N-t-butyl hydronitroxide and benzaldehyde (11-13). PBN,
as purchased, often contains N-t-butyl hydroxylamine (14). Benzaldehyde,
is both mutagenic (15) and carcinogenic (16). N-t-butyl hydroxylamine is
a primary hydroxylamine that can be oxidized, under certain conditions
(such as with UV or Fe+3), to N-(t-butyl)aminoxyl (also referred as
N-t-butyl hydronitroxide (10-12). N-(t-butyl)aminoxyl and the
corresponding N-hydroxylamine are primary amines and are thus different
from the well known cyclic-nitroxides/cyclic-hydroxylamines. The
antioxidative and protective features of some
cyclic-nitroxides/cyclic-hydroxylamines are known. Probably the most
important feature in this regard, is their ability to catalyze superoxide
radical dismutation to form H2O2 (17-21). In vitro
cyclic-nitroxides can either be oxidized to oxo-ammonium cation or
reduced to the corresponding hydroxylamine by superoxide radical,
depending on the type of cyclic-nitroxide. Thus cyclic-hydroxylamine or
the corresponding oxo-ammonium cation are intermediates during the
dismutation of superoxide radical by nitroxide. Interestingly, the
oxo-ammonium cation species is reduced to the corresponding
cyclic-hydroxylamine by the cellular reductant NADH, which suggests that
cyclic-hydroxylamine can be the dominant form inside the cells. In
addition the cyclic-nitroxide species can undergo one electron reduction
to the corresponding cyclic-hydroxylamine, a reaction proposed to be
mediated by mitochondrial coenzyme Q and ascorbic acid (21-23).
Mitochondrial cytochrome c oxidase can also oxidize the
cyclic-hydroxylamine to the corresponding nitroxide (24). Thus, it
appears that mitochondria can contribute to the cycling of
cyclic-nitroxides/cyclic-hydroxylamines, which in turn can facilitate
dismutation of superoxide radical to H2O2. The N-t-butyl
hydroxylamine and the other N-hydroxylamines tested in this study are
primary N-hydroxylamines which have not been previously examined as
antioxidants.

[0009]The pharmaceutical compositions generally comprise a pharmaceutical
composition comprising an orally administrable effective unit solid
dosage of a primary N-hydroxylamine or a pharmaceutically acceptable salt
thereof and substantially free of a nitrone corresponding to the
hydroxylamine, wherein the hydroxylamine has the formula:

[0011]The invention provides more specific aspects of this embodiment:

[0012]wherein at least one R of R1, R2 and R3 is selected
from unsubstituted (C0-C10) alkyl, alkenyl and alkynyl;

[0013]wherein at least one R of R1, R2 and R3 is selected
from unsubstituted (C0-C18) alkyl, cycloalkyl, alkenyl and alkynyl, and
the R is selected from: CH3--(CH2)n1,
(CH3--(CH2).sub.n2--)2 CH,
(CH3--(CH2).sub.n2--)3, cyclopentyl, cyclohexyl,
(CH2═CH--CH2)n3 and (CH═C--CH2--)n3,
wherein n1=1 to 18, n2=1 to 17 and n3=1 to 3;

[0014]wherein at least one R of R1, R2 and R3 is selected
from unsubstituted (C0-C10) alkyl, alkenyl and alkynyl, and the
hydroxylamine is selected from:

[0018]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) oxyl;

[0019]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) oxyl and the R is selected from:
hydroxyl, hydroxyalkyl (HO--(CH2)n1), hydroxyaryl selected from
benzylalcohol, phenol and naphthol, alkoxy (O--(CH2)n1) and
aryloxy selected from phenoxy, benzyloxy and naphthyloxy, wherein n1=1 to
18;

[0020]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18)alkyl hydroxyl or arylhydroxyl and
the hydroxylamine is selected from:

[0040]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted alkyl (C0-C18) or aryl amino;

[0041]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted alkyl (C0-C18) or aryl amino and the R is
selected from primary alkyl amine selected from methylamine, ethylamine,
propylamine, butylamine and hexylamine, secondary amine selected from
dimethylamine, diethylamine and dipropylamine, tertiary amine selected
from trimethyl and trietylamine, and quaternary amine selected from
tetramethyl and tetra-ethylammonium salts;

[0042]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted alkyl(C0-C18) or aryl amine and the
hydroxylamine is selected from: [0043]N-aminomethylhydroxylamine,
[0044]N-(2-aminoethyl)hydroxylamine,
[0045]N--(N-methylamino)methylhydroxylamine,
[0046]N--(N,N-dimethylamino)methylhydroxylamine,
[0047]N--(N,N,N-trimethylammonium)methylhydroxylamine,
[0048]N-(3-aminopropyl)hydroxylamine,
[0049]N-(6-aminohexyl)hydroxylamine,
[0050]N-(4-aminobenzyl)hydroxylamine,
[0051]Hydroxylamine-1-methylpyridinium and
[0052]Hydroxylamine-1-methylquinolinium;

[0053]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) alkyl or aryl nitro;

[0054]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted alkyl(C0-C18) or aryl nitro and the R is
selected from alkylnitro selected from nitromethyl, nitroethyl,
nitropropyl, nitrobutyl, nitropentyl, nitrohexyl and nitrobenzyl, and
arylnitro selected from nitrophenyl and nitronaphthyl;

[0055]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted alkyl (C0-C18) or aryl nitro and the
hydroxylamine is selected from:

[0056]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) nitroso;

[0057]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) nitroso and the R is selected from
aliphatic nitrosamines and aromatic nitroso;

[0058]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted nitroso (C0-C18) and the hydroxylamine is
selected from: [0059]N--(N-methyl-N-nitroso-amino)methyl hydroxylamine,
[0060]N--(N-methyl-N-nitroso-2-amino)ethylhyroxylamine,
[0061]N--(N-methyl-N-nitroso-3-amino)propylhydroxylamine and
[0062]N-(p-nitroso)benzylhydroxylamine;

[0063]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted oxime;

[0064]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) oxime and the R is selected from:
acetaldoxime, propionaldoxime, butanaldoxime and benzaldoxime;

[0065]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted oxime (C0-C18) and the hydroxylamine is
selected from:

[0066]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C10) hydrazone;

[0067]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C10) hydrazone and the R is selected
from: acetaldehyde hydrazone, propanaldehyde hydrazone, butanaldehyde
hydrazone and phenylhydrazine;

[0068]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted hydrazone (C0-C10) and the hydroxylamine is
selected from:

[0069]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted azo;

[0070]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted azo and the R is selected from: azobenzene,
p-(phenylazo)benzyl and p-diazobenzyl;

[0071]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted azo and the hydroxylamine is selected from:
[0072]N-(p-phenylazo)benzylhydroxylamine,
[0073]N-(p-diazobenzyl)hydroxylamine and
[0074]N-(p-methoxylphenylazo)benzylhydroxylamine

[0075]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) thiol;

[0076]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) thiol and the R is selected from
(C0-C18) alkylthiol selected from methyl, ethyl, propyl, butyl, pentyl
and hexyl thiol, and arylthio selected from thiophenol and benzylthiol;

[0077]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) thiol and the hydroxylamine is
selected from:

[0078]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) sulfonic acid;

[0079]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) sulfonic acid and the R is selected
from methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid,
butanesulfonic acid and p-toluenesulfonic acid;

[0080]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) sulfonic acid and the hydroxylamine
is selected from:

[0084]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted hydroxylamine;

[0085]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted hydroxylamine and R is selected from
N-methylhydroxylamine, N-ethylhyroxylamine, N-propylhydroxylamine
N-butylhydroxylamine, N-pentylhydroxylamine, and N-benzylhydroxylamine;

[0086]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted hydroxylamine and the hydroxylamine is
selected from:

[0087]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) phosphoester;

[0088]wherein at least one R of R1, R2 and R3 is
substituted or unsubstituted (C0-C18) phosphoester and the R is selected
from: dimethylphosphate, diethylphosphate, dipropylphosphate and
benzylphosphate;

[0105]wherein the nitrone is less than 10%, 1%, 0.1% (wt/wt) of the
hydroxylamine in the composition;

[0106]wherein the composition is packaged with a label identifying the
primary N-hydroxylamine and prescribing a pharmaceutical use thereof,
particularly wherein the use is other than oncological and/or comprises
reducing oxidative damage or delaying senescence; and

[0107]wherein the composition further comprising an effective amount of a
carnitine.

[0108]The invention also provides a wide variety of methods of using
primary N-hydroxylamines, including the subject hydroxylamines,
including:

[0109]a method for reducing oxidative damage to, or delaying senescence of
a cell comprising the steps of: identifying a cell as subject to or at
risk of undesirable oxidative damage or senescence; and contacting the
cell with a composition comprising an effective amount of a primary
hydroxylamine and substantially free of a nitrone corresponding to the
hydroxylamine, particularly, wherein the cell is contained in other than
a cancerous host;

[0110]a method for screening for primary N-hydroxylamines which reduce
oxidative damage to, or delay senescence of cells, comprising the steps
of: contacting cells with a candidate primary N-hydroxylamine under
conditions whereby, but for the presence of the hydroxylamine, the cells
present a reference amount of oxidative damage or senescence; detecting
post-treatment amounts of oxidative damage or senescence of the cells;
wherein a lesser amount of post-treatment than reference amounts of
oxidative damage or senescence indicates that the hydroxylamine reduces
oxidative damage or delays senescence of the cells;

[0111]a method for improving short term memory in a patient, said method
comprising administering to said patient a pharmaceutical composition
comprising an effective short term memory improving amount of a subject
hydroxylamine;

[0112]a method for treating a patient with an acute central nervous system
disorder, said method comprising administering to said patient a
pharmaceutical composition comprising an effective acute central nervous
system disorder-treating amount of a subject hydroxylamine, particularly
wherein the acute central nervous system disorder treated is stroke;

[0113]a method for treating a patient with an acute cardiovascular
disorder, said method comprising administering to said patient a
pharmaceutical composition comprising an effective acute cardiovascular
disorder-treating amount of a subject hydroxylamine, particularly wherein
the acute cardiovascular disorder treated is cardiac infarction;

[0114]a method for treating a patient with a neurodegenerative disease
which method comprises administering to said patient a pharmaceutical
composition comprising an effective neurodegenerative disease-treating
amount of a subject hydroxylamine, and a method for preventing the onset
of a neurodegenerative disease in a patient at risk for developing the
neurodegenerative disease which method comprises administering to said
patient a pharmaceutical composition comprising an effective
neurodegenerative disease-preventing amount of a subject hydroxylamine,
particularly wherein the neurodegenerative disease treated and/or
prevented in the above methods is Alzheimer's disease, Parkinson's
disease, HIV dementia and the like;

[0115]a method for treating a patient with an autoimmune disease which
method comprises administering to said patient a pharmaceutical
composition comprising an effective autoimmune disease-treating amount of
a subject hydroxylamine, and a method for preventing the onset of an
autoimmune disease in a patient at risk for developing the autoimmune
disease which method comprises administering to said patient a
pharmaceutical composition comprising an effective autoimmune
disease-preventing amount of a subject hydroxylamine, particularly
wherein the autoimmune disease treated and/or prevented in the above
methods is systemic lupus, multiple sclerosis and the like;

[0116]a method for treating a patient with an inflammatory disease which
method comprises administering to said patient a pharmaceutical
composition comprising an effective inflammatory disease-treating amount
of a subject hydroxylamine, and a method for preventing the onset of an
inflammatory disease in a patient at risk for developing the inflammatory
disease which method comprises administering to said patient a
pharmaceutical composition comprising an effective inflammatory
disease-preventing amount of a subject hydroxylamine, particularly
wherein the inflammatory disease treated and/or prevented in the above
methods is rheumatoid arthritis, septic shock, erythema nodosum
leprosyia, uveitis and the like.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0117]The following descriptions of particular embodiments and examples
are offered by way of illustration and not by way of limitation.

DEFINITIONS

[0118]Unless contraindicated or noted otherwise, in these descriptions and
throughout this specification, the terms "a" and "an" mean one or more,
the term "or" means and/or.

[0119]Alkyl" refers to monovalent alkyl groups preferably having from 1 to
about 18 carbon atoms, more preferably 1 to 8 carbon atoms and still more
preferably 1 to 6 carbon atoms. This term is exemplified by groups such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl,
n-hexyl, n-octyl, tert-octyl and the like. The term "lower alkyl" refers
to alkyl groups having 1 to 6 carbon atoms.

[0120]Substituted alkyl" refers to an alkyl group preferably having from 1
to about 12 carbon atoms, more preferably 1 to 8 carbon atoms and still
more preferably 1 to 6 carbon atoms, which is substituted, preferably
with, from 1 to 3 substituents selected from the group consisting of
alkoxy, amino, mono- and dialkylamino, aminoacyl, amido, alkoxycarbonyl,
aryl, carboxyl, cyano, halo, heterocyclic, hydroxy, nitro, thioalkoxy and
the like.

[0121]Alkenyl" refers to alkenyl groups preferably having from 2 to 10
carbon atoms and more preferably 2 to 6 carbon atoms and having at least
1 and preferably from 1-2 sites of alkenyl unsaturation. Preferred
alkenyl groups include ethenyl (--CH═CH2), n-propenyl (--CH2
CH═CH2), isopropenyl (--C(CH3)═CH2), and the like.

[0122]Alkynyl" refers to alkynyl groups preferably having from 2 to 10
carbon atoms and more preferably 2 to 6 carbon atoms and having at least
1 and preferably from 1-2 sites of alkynyl unsaturation. Preferred
alkynyl groups include ethynyl (--C≡CH), propargyl (--CH2C═CH),
and the like.

[0123]Alkcycloalkyl" refers to -alkylene-cycloalkyl groups preferably
having from 1 to 10 carbon atoms in the alkylene moiety and from 3 to 8
carbon atoms in the cycloalkyl moiety. Such alkcycloalkyl groups are
exemplified by --CH2-cyclopropyl, --CH2-cyclopentyl, --CH2CH2-cyclohexyl,
and the like.

[0125]Alkoxycarbonyl" refers to the group --C(O)OR where R is alkyl.
"Aminocarbonyl" refers to the group --C(O)NRR where each R is
independently hydrogen or alkyl.

[0126]Aminoacyl" refers to the group --NRC(O)R where each R is
independently hydrogen or alkyl.

[0127]Aryl" refers to an unsaturated aromatic carbocyclic group of from 6
to 14 carbon atoms having a single ring (e.g., phenyl) or multiple
condensed rings (e.g., naphthyl or anthryl). Preferred aryls include
phenyl, naphthyl and the like. Unless otherwise constrained by the
definition for the individual substituent, such aryl groups can
optionally be substituted, preferably with from 1 to 3 substituents
selected from the group consisting of alkyl, substituted alkyl, alkoxy,
alkenyl, alkynyl, amino, aminoacyl, aminocarbonyl, alkoxycarbonyl, aryl,
carboxyl, cyano, halo, hydroxy, nitro, trihalomethyl and the like.

[0128]Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon
atoms having a single cyclic ring or multiple condensed rings which can
be optionally substituted with from 1 to 3 alkyl groups. Such cycloalkyl
groups include, by way of example, single ring structures such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl,
2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring
structures such as adamantanyl, and the like.

[0129]Cycloalkenyl" refers to cyclic alkenyl groups of from 4 to 10 carbon
atoms having a single cyclic ring and at least one point of internal
unsaturation which can be optionally substituted with from 1 to 3 alkyl
groups. Examples of suitable cycloalkenyl groups include, for instance,
cyclopent-3-enyl, cyclohex-2-enyl, cyclooct-3-enyl and the like.

[0130]Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.
Preferred halo groups are either fluoro or chloro.

[0131]Thioalkoxy" refers to the group "alkyl-S-". Preferred thioalkoxy
groups include, by way of example, thiomethoxy, thioethoxy,
n-thiopropoxy, isothiopropoxy, n-thiobutoxy and the like.

[0132]Heterocycle" or "heterocyclic" refers to a monovalent saturated or
unsaturated group having a single ring or multiple condensed rings, from
1 to 10 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen,
sulfur or oxygen within the ring. Examples of heterocycles include, but
are not limited to, morpholine, piperazine, imidazolidine, pyrrolidine,
piperidine and the like.

[0133]Pharmaceutically acceptable salt" refers to pharmaceutically
acceptable salts which are derived from a variety of organic and
inorganic counter ions well known in the art and include, by way of
example only, sodium, potassium, calcium, magnesium, ammonium,
tetraalkylammonium, and the like; and when the molecule contains a basic
functionality, salts of organic or inorganic acids, such as
hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate and the
like. Pharmaceutically acceptable salts of the hydroxylamines of this
invention are prepared using conventional procedures well known to those
skilled in the art including, for example, treating a sulfonic acid
derivative with an appropriate base.

[0134]A corresponding nitrone means a nitrone condensate of the
hydroxylamine and hence, having the same nitrogen bound R group, i.e. the
condensation product of the primary N-hydroxyl amine with an aldehyde.
Substantially free of a corresponding nitrone means the nitrone is less
than 10%, preferably less than 1%, more preferably less than 0.1% (wt/wt)
of the corresponding hydroxylamine in the composition.

[0135]Orally administrable means both safe and effective when orally
administered.

General Synthetic Procedures

[0136]The hydroxylamine compounds of this invention can be purchased
commercially and/or prepared from readily available starting materials
using conventional methods and procedures. It will be appreciated that
where typical or preferred process conditions (i.e., reaction
temperatures, times, mole ratios of reactants, solvents, pressures, etc.)
are given, other process conditions can also be used unless otherwise
stated. Optimum reaction conditions may vary with the particular
reactants or solvent used, but such conditions can be determined by one
skilled in the art by routine optimization procedures.

[0137]The hydroxylamine may often be prepared by reduction of the
corresponding nitro compound using a suitable catalyst such as an
activated zinc/acetic acid catalyst or an aluminum/mercury amalgam
catalyst. This reaction is typically conducted at a temperature ranging
from about 15° C. to about 100° C. for about 0.5 to 12
hours, preferably about 2 to 6 hours, in an aqueous reaction media, such
as an alcohol/water mixture in the case of the zinc catalyst or an
ether/water mixture in the case of the aluminum amalgam catalyst.
Hydroxylamines can also be prepared by reduction of oximes with hydride
reducing agents, such as sodium cyanoborohydride. Aliphatic nitro
compounds (in the form of their salts) can also be reduced to
hydroxylamines. Since some hydroxylamines have limited stability, such
compounds are generally prepared immediately prior to reaction with a
carbonyl compound. Alternatively, hydroxylamines can often be stored (or
purchased commercially) as their hydrochloride salts. In such cases, the
free hydroxylamine is typically generated immediately prior to reaction
with a carbonyl compound by reaction of the hydrochloride salt with a
suitable base, such as sodium hydroxide, sodium methoxide and the like.

[0138]In a particular embodiment, at least one of R1, R2 and
R3 is selected from the group consisting of alkyl, substituted
alkyl, alkenyl, alkynyl, alkaryl, aryl, alkcycloalkyl, cycloalkyl and
cycloalkenyl; particularly wherein R1 is selected from the group
consisting of alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aryl,
alkcycloalkyl, cycloalkyl and cycloalkenyl and R2 and R3 are H.
Preferred hydroxylamines of this invention include, but are not limited
to, N-methylhydroxylamine, N-ethylhyroxylamine, N-n-propylhydroxylamine,
N-isopropylhydroxylamine, N-n-butylhydroxylamine,
N-isobutylhydroxylamine, N-sec-butylhydroxylamine,
N-tert-butylhydroxylamine, N-n-pentylhydroxylamine,
N-cyclopentylhydroxylamine, N-n-hexylhydroxylamine,
N-cyclohexylhydroxylamine, N-n-octylhydroxylamine,
N-tert-octylhydroxylamine, N-phenylhydroxylamine and the like. Also
included are compounds having multiple primary hydroxylamine moieties,
e.g. methyl di(hydroxylamine).

[0139]In some cases, the hydroxylamines of this invention will contain one
or more chiral centers. Typically, such compounds will be prepared as a
racemic mixture. If desired, however, such compounds can be prepared or
isolated as pure stereoisomers, i.e., as individual enantiomers or
diastereomers, or as stereoisomer-enriched mixtures. All such
stereoisomers (and enriched mixtures) of the disclosed hydroxylamines are
included within the scope of this invention. Pure stereoisomers (or
enriched mixtures) may be prepared using, for example, optically active
starting materials or stereoselective reagents well known in the art.
Alternatively, racemic mixtures of such compounds can be separated using,
for example, chiral column chromatography, chiral resolving agents and
the like.

Pharmaceutical Compositions

[0140]When employed as pharmaceuticals, the hydroxylamines of this
invention are typically administered in the form of a pharmaceutical
composition comprising at least one active hydroxylamine compound and a
carrier, vehicle or excipient suitable for use in pharmaceutical
compositions. Without being limited thereto, such materials include
diluents, binders and adhesives, lubricants, plasticizers, disintegrants,
colorants, bulking substances, flavorings, sweeteners and miscellaneous
materials such as buffers and adsorbents in order to prepare a particular
medicated composition. Such carriers are well known in the pharmaceutical
art as are procedures for preparing pharmaceutical compositions.

[0142]The dosage forms may include a variety of other ingredients,
including binders, solvents, bulking agents, plasticizers etc. Binders
may be selected from a wide range of materials such as
hydroxypropylmethylcellulose, ethylcellulose, or other suitable cellulose
derivatives, povidone, acrylic and methacrylic acid co-polymers,
pharmaceutical glaze, gums, milk derivatives, such as whey, starches and
derivatives, as well as other conventional binders well known to persons
skilled in the art. Exemplary non-limiting solvents are water, ethanol,
isopropyl alcohol, methylene chloride or mixtures and combinations
thereof. Exemplary non-limiting bulking substances include sugar,
lactose, gelatin, starch, and silicon dioxide. The plasticizers used in
the dissolution modifying system are preferably previously dissolved in
an organic solvent and added in solution form. Preferred plasticizers may
be selected from the group consisting of diethyl phthalate, diethyl
sebacate, triethyl citrate, crotonic acid, propylene glycol, butyl
phthalate, dibutyl sebacate, castor oil and mixtures thereof, without
limitation. As is evident, the plasticizers may be hydrophobic as well as
hydrophilic in nature. Water-insoluble hydrophobic substances, such as
diethyl phthalate, diethyl sebacate and castor oil are used to delay the
release of water-soluble drugs, such as potassium chloride. In contrast,
hydrophilic plasticizers are used when water-insoluble drugs are employed
which aid in dissolving the encapsulating film, making channels in the
surface, which aid in drug release.

[0143]A wide variety of orally administrable compositions may be used. In
a particular embodiment, the oral compositions are provided in solid
discrete, self-contained dosage units, such as tablets, caplets,
lozenges, capsules, gums, etc., which may comprise or be filled with
liquid or solid dosage of the hydroxylamine. A wide variety of dosages
may be used, depending on the application and empirical determination;
typical dosages range from 10 ug to 1 g, preferably at least 100 ug, more
preferably at least 1 mg, more preferably at least 10 mg, most preferably
at least 100 mg.

[0144]The compositions for oral administration can take the form of bulk
liquid solutions or suspensions, or bulk powders. More commonly, however,
the compositions are presented in unit dosage forms to facilitate
accurate dosing. The term "unit dosage forms" refers to physically
discrete units suitable as unitary dosages for human subjects and other
mammals, each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in association with
a suitable pharmaceutical excipient. Typical unit dosage forms include
prefilled, premeasured ampules or syringes of the liquid compositions or
pills, tablets, capsules or the like in the case of solid compositions.
In such compositions, the hydroxylamine compound is usually a minor
component (from about 0.1 to about 50% by weight or preferably from about
1 to about 40% by weight) with the remainder being various vehicles or
carriers and processing aids helpful for forming the desired dosing form.

[0145]Liquid forms suitable for oral administration may include a suitable
aqueous or nonaqueous vehicle with buffers, suspending and dispensing
agents, colorants, flavors and the like. Solid forms may include, for
example, any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating agent
such as alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate; a glidant such as colloidal silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such
as peppermint, methyl salicylate, or orange flavoring.

[0146]Injectable compositions are typically based upon injectable sterile
saline or phosphate-buffered saline or other injectable carriers known in
the art. As before, the hydroxylamine compound in such compositions is
typically a minor component, often being from about 0.05 to 10% by weight
with the remainder being the injectable carrier and the like.

[0147]The above described components for orally administrable or
injectable compositions are merely representative. Other materials as
well as processing techniques and the like are set forth in Part 8 of
Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing
Company, Easton, Pa., which is incorporated herein by reference.

[0148]The dosage forms of the present invention involve the administration
of an active therapeutic substance or multiple active therapeutic
substances in a single dose during a 24 hour period of time or multiple
doses during a 24 hour period of time. The doses may be uneven in that
each dose is different from at least one other dose.

[0149]The subject compositions may be administered to effect various forms
of release, which include, without limitation, immediate release,
extended release, controlled release, timed release, sustained release,
delayed release, long acting, pulsatile delivery, etc., using well known
procedures and techniques available to the ordinary skilled artisan. A
description of representative sustained release materials can be found in
the incorporated materials in Remington's Pharmaceutical Sciences. For
example, prodrugs which liberate the active compound in vivo by enzymatic
or hydrolytic cleavage may be used. The following formulation examples
illustrate representative pharmaceutical compositions of this invention.
The present invention, however, is not limited to the following
exemplified pharmaceutical compositions.

[0150]Formulation 1--Tablets: A compound (e.g. tert-buytlhydroxylamine) is
admixed as a dry powder with a dry gelatin binder in an approximate 1:2
weight ratio. A minor amount of magnesium stearate is added as a
lubricant. The mixture is formed into 240-270 mg tablets (80-90 mg of
active hydroxylamine compound per tablet) in a tablet press.

[0151]Formulation 2--Capsules: A compound (e.g. tert-buytlhydroxylamine)
is admixed as a dry powder with a starch diluent in an approximate 1:1
weight ratio. The mixture is filled into 250 mg capsules (125 mg of
active hydroxylamine compound per capsule).

[0152]Formulation 3--Liquid: A compound (e.g. tert-buytlhydroxylamine) (50
mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through
a No. 10 mesh U.S. sieve, and then mixed with a previously made solution
of microcrystalline cellulose and sodium carboxymethyl cellulose (11:89,
50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted
with water and added with stirring. Sufficient water is then added to
produce a total volume of 5 mL.

[0153]Formulation 4--Tablets: The compound (e.g. tert-buytlhydroxylamine)
is admixed as a dry powder with a dry gelatin binder in an approximate
1:2 weight ratio. A minor amount of magnesium stearate is added as a
lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of
active hydroxylamine compound) in a tablet press.

[0154]Formulation 5--Injection: The compound (e.g.
tert-buytlhydroxylamine) is dissolved in a buffered sterile saline
injectable aqueous medium to a concentration of approximately 5 mg/ml.

[0161]As therapeutics and/or prophylactics, the hydroxylamines of this
invention have been found to be useful for treating a wide variety of
medical dysfunctions and diseases, in humans and animal. Among the
various medical conditions which may be prevented and/or treated, the
hydroxylamines of this invention are particularly useful for treating
conditions involving acute oxidate damage, such as acute intense
oxidative damage to a region of the central nervous system, e.g. stroke,
conditions associated with stroke, concussion and subarachnoid hemorrhage
or chronic oxidate damage, such as is associated with senescence and
aging. Accordingly, the subject compositions are useful in treating a
variety of dysfunctions or disorders characterized by oxidized proteins,
nucleic acids or lipids in the tissues, cells, or associated fluids (such
as the blood). Cellular, tissue, systemic and organismal indicia of
oxidative damage are known in the art and exemplified below; for example,
in vitro cellular oxidative damage and senescence may be measured as
described in Chen et al. (1995) Proc. Natl. Acad. Sci. USA 92, 4337-4341.

[0162]Disorders are generally divided into disorders of the central and
peripheral nervous system and disorders of the peripheral organs.
Disorders of the CNS include stroke, aging, neurodegenerative conditions,
such as Alzheimer's disease, Parkinsonism, concussion, aneurysm,
ventricular hemorrhage and associated vasospasm, migraine and other
vascular headaches, spinal cord trauma, neuroanesthesia adjunct,
HIV-dementia and the like. Disorders of the peripheral nervous system
include diabetic peripheral neuropathy and traumatic nerve damage.
Peripheral organ disease includes atherosclerosis (both diabetic and
spontaneous), chronic obstructive pulmonary disease (COPD), pancreatitis,
pulmonary fibrosis due to chemotherapeutic agents, angioplasty, trauma,
burns, ischemic bowel disease, wounds, ulcers and bed sores, lupus,
ulcerative colitis, organ transplantation, renal hypertertsion,
overexertion of skeletal muscle, epistaxis (pulmonary bleeding),
autoimmune conditions, such as systemic lupus (erythematosus), multiple
sclerosis and the like; and inflammatory conditions, such as inflammatory
bowel disease, rheumatoid arthritis, septic shock, erythema nodosum
leprosy, septicemia, uveitis, and the like. With regard to these disease
classifications, it will be appreciated by those skilled in the art, that
some disease conditions may be classified as, for example, both
autoimmune and inflammatory conditions, such as multiple sclerosis and
the like.

[0163]Other conditions associated: with excessive oxidation of proteins or
lipids that can be treated include undesirable or altered oxidation of
low density lipoprotein, and dysfunction from exposure to radiation,
including x-ray, ultraviolet, gamma and beta radiation, and cytotoxic
compounds, including those used for chemotherapy for cancer and viral
infections.

[0164]Accordingly, in one of its method aspects, this invention provides a
method for treating a patient with an acute central nervous system
disorder, said method comprising administering to said patient a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and an effective acute central nervous system disorder-treating
subject hydroxylamine. In a preferred embodiment of this method, the
acute central nervous system disorder treated is stroke.

[0165]In another of its method aspects, this invention provides a method
for treating a patient with an acute cardiovascular disorder, said method
comprising administering to said patient a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective acute
cardiovascular disorder-treating amount of a subject hydroxylamine. In a
preferred embodiment of this method, the acute cardiovascular disorder
treated is cardiac infarction.

[0166]In still another of its method aspects, this invention is directed
to a method for treating a patient with a neurodegenerative disease which
method comprises administering to said patient a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and an
effective neurodegenerative disease-treating amount of a subject
hydroxylamine. Additionally, this invention is directed to a method for
preventing the onset of a neurodegenerative disease in a patient at risk
for developing the neurodegenerative disease which method comprises
administering to said patient a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and an effective neurodegenerative
disease-preventing amount of a subject hydroxylamine. In preferred
embodiments of this invention, the neurodegenerative disease treated
and/or prevented in the above methods is Alzheimer's disease, Parkinson's
disease, HIV dementia, a dopamine-associated neurodegenerative condition
and the like.

[0167]In yet another of its method aspects, this invention is directed to
a method for treating a patient with an autoimmune disease which method
comprises administering to said patient a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective
autoimmune disease-treating amount of a subject hydroxylamine. This
invention is also directed to a method for preventing the onset of an
autoimmune disease in a patient at risk for developing the autoimmune
disease which method comprises administering to said patient a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and an effective autoimmune disease-preventing amount of a
subject hydroxylamine. In preferred embodiments of this invention, the
autoimmune disease treated and/or prevented in the above methods is
systemic lupus, multiple sclerosis and the like.

[0168]In still another of its method aspects, this invention is directed
to a method for treating a patient with an inflammatory disease which
method comprises administering to said patient a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and an
effective inflammatory disease-treating amount of a subject
hydroxylamine. Additionally, this invention is directed to a method for
preventing the onset of an inflammatory disease in a patient at risk for
developing the inflammatory disease which method comprises administering
to said patient a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and an effective inflammatory
disease-preventing amount of a subject hydroxylamine: In preferred
embodiments of this invention, the inflammatory disease treated and/or
prevented in the above methods is rheumatoid arthritis, septic shock,
erythema nodosum leprosy, septicemia, uveitis and the like.

[0169]In another aspect this invention provides a method for treating a
patient suffering from a condition characterized by progressive loss of
nervous system function due to mitochondrial dysfunction. This method
involves administering to the patient with loss of central nervous system
function an effective amount of one or more of the pharmaceutical
compositions just described.

[0170]In each aspect, the invention may be implemented by a first
diagnostic step, e.g. determining that the patient is suffering from,
subject to, or predisposed to a target disease or condition followed by
prescribing and/or administering to the patient a subject hydroxylamine,
optionally followed by a evaluation/confirmation/prognosis step, e.g.
determining an effect of the treatment, such as an amelioration of
symptom of a targeted disease or condition or an indicator thereof.

Administration

[0171]The subject compositions may be formulated for administration by any
route, including without limitation, oral, buccal, sublingual, rectal,
parenteral, topical, inhalational, including intranasal, injectable,
including subcutaneous, intravenous, intramuscular, etc., topical,
including transdermal, etc. The subject compositions are administered in
a pharmaceutically (including therapeutically, prophylactically and
diagnostically) effective amount. The amount of the compound actually
administered will typically be determined by a physician, in the light of
the relevant circumstances, including the condition to be treated, the
chosen route of administration, the actual compound administered, the
age, weight, and response of the individual patient, the severity of the
patient's symptoms, and the like.

[0172]Intravenous dose levels for treating acute medical conditions range
from about 0.1 mg/kg/hour to at least 10 mg/kg/hour over a period of from
about 1 to about 120 hours and especially 24 to 96 hours. Preferably, an
amount of at least about 0.2 mg/kg/hour is administered to the patient. A
preloading bolus of from about 10 mg to about 500 mg may also be
administered to achieve adequate steady state levels. While intravenous
administration is preferred for acute treatments, other forms of
parenteral administration, such as intramuscular injection can be used,
as well. In such cases, dose levels similar to those described above may
be employed.

[0173]Another acute condition which can be advantageously treated with the
hydroxylamines of this invention is acute oxidative damage to the
cardiovascular system, such as the damage which occurs in a patient who
has suffered a cardiac infarction or the like. When treating such a
condition, a pharmaceutical composition comprising a hydroxylamine is
administered parenterally, e.g. intravenously, at doses similar to those
described above for stroke and other acute CNS conditions.

[0174]As discussed above, the compounds described herein are suitable for
use in a variety of drug delivery systems. Injection dose levels for
treating neurodegenerative, autoimmune and inflammatory conditions range
from about 0.1 mg/kg/hour to at least 10 mg/kg/hour, all for from about 1
to about 120 hours and especially 24 to 96 hours. A preloading bolus of
from about 0.1 mg/kg to about 10 mg/kg or more may also be administered
to achieve adequate steady state levels. The maximum total dose is not
expected to exceed about 2 g/day for a 40 to 80 kg human patient.

[0175]For the prevention and/or treatment of long-term conditions, such as
neurodegenerative and autoimmune conditions, the regimen for treatment
usually stretches over many months or years so oral dosing is preferred
for patient convenience and tolerance. With oral dosing, one to five and
especially two to four and typically three oral doses per day are
representative regimens. Using these dosing patterns, each dose provides
from about 0.02 to about 50 mg/kg of hydroxylamine, with preferred doses
each providing from about 0.04 to about 30 mg/kg and especially about 1
to about 10 mg/kg.

[0176]When used to prevent the onset of a degenerative condition, such as
a neurodegenerative, autoimmune or inflammatory condition, the
hydroxylamine compounds of this invention will be administered to a
patient at risk for developing the condition, typically on the advice and
under the supervision of a physician, at the dosage levels described
above. Patients at risk for developing a particular condition generally
include those that have a family history of the condition, or those who
have been identified by genetic testing or screening to be particularly
susceptible to developing the condition. When used prophylactically, a
pharmaceutical composition comprising a hydroxylamine is administered
orally to the predisposed patient. The doses for this oral therapy will
typically be the same as those set forth above for treating persons
suffering from the neurodegenerative, autoimmune or inflammatory
condition.

[0177]The compounds of this invention can be administered as the sole
active agent or they can be administered in combination with other
agents, including other active hydroxylamine compounds.

[0178]The following synthetic and biological examples are offered to
illustrate this invention and are not to be construed in any way as
limiting the scope of this invention.

EXAMPLES

Example 1

[0179]Synthesis of N-Isopropylhydroxylamine. Acetic acid (10.8 g) was
added to a cooled solution of 2-nitropropane (5.35 g) and zinc dust (5.89
g) in 95% ethanol (350 mL) at such a rate to maintain the temperature
below 10° C. The reaction was stirred for three hours and the
solvent removed in vacuo. The residue was extracted three times with
dichloromethane. The combined extracts were dried over magnesium sulfate,
filtered, and solvent stripped. The crude hydroxylamine product was used
without further purification. Other hydroxylamines may also be prepared
by this procedure.

Example 2

[0180]Synthesis of Hydroxylamines By Reduction of Oximes. Various
hydroxylamines were prepared according to the procedures of R. F. Borch
et al., J. Amer. Chem. Soc., 1971, 93(3):2897 from the corresponding
oxime. Specifically, a 3-necked round bottom flask equipped with a
stirring motor, a pH meter probe and an addition funnel is charged with a
solution of the oxime in methanol (ca. 0.4M). To the stirring solution is
added 0.68 equivalents of NaBH3 CN in portions. The addition funnel
is filled with 4M HCl in MeOH. The amount of the acid solution prepared
should be roughly 3/4 of the volume of MeOH used to dissolve the oxime.
The HCl solution is then added slowly to the oxime until pH comes down to
about 4 and stabilizes at that value. The solution is then allowed to
stir at ambient temperature for ca. 4 hours. HCl is added as necessary to
keep the pH at 4. (A small sample can be periodically removed and
worked-up to determine if the reaction is complete). When the reaction is
complete, the solution is decanted into a 1-necked round bottom flask and
MeOH is removed in vacuo. (While removing the methanol by
rotoevaporation, the solvent trap should be filled with NaOH (1 eq.) to
quench HCN stripped off with MeOH). After the methanol has been removed,
the residue is dissolved in water and extracted with methylene chloride
(4×). The organic phases are combined, dried over MgSO4 and
stripped to dryness to provide the hydroxylamine product (as determined
by NMR and DSC).

Example 3

[0181]Synthesis of N-Cyclohexylhydroxylamine. N-Cyclohexylhydroxylamine
hydrochloride (commercially available from Aldrich, 1001 West Saint Paul
Avenue, Milwaukee, Wis. 53233 U.S.A.) was suspended in ether (about 200
mL of ether for 6 grams of the hydroxylamine salt) and extracted three
times with 5% NaOH in brine. The organic phase (white fluffy crystals of
N-cyclohexylhydroxylamine suspended in ether) was transferred to a round
bottom flask and the ether was removed in vacuo. The resulting crystals
were dried under a high vacuum for about 20 min. to afford the title
compound.

Example 4

[0182]Treatment of Acute CNS Disorders. In this example, the ability of
subject hydroxylamines to reduce the infarct volume in an in vivo stroke
model is demonstrated. A rat permanent middle cerebral artery occlusion
(MCAO) model is used to determine stroke treatment efficacy. MCAO is a
representative model of acute CNS disorders. See, for example, M. D.
Ginsberg et al., "Rodent Models of Cerebral Ischemia" (1989) Stroke,
20:1627-1642. In this stroke model, the middle cerebral artery is
permanently occluded via cauterization to produce a focal stroke. The
hydroxylamines are then administered as a 10 mg/kg i.v. bolus dose three
hours post MCAO through a catheter surgically implanted in the jugular
vein. Two days post MCAO, the rats are sacrificed and the extent of brain
damage assessed using tetrazolium staining (TTC staining) followed by
computer image analysis to quantitate infarct volumes, i.e., the regions
of dead tissue. The mean infarct volume for rats treated with the test
compound is significantly less than the mean infarct volume for control
rats not treated with the hydroxylamine. Thus, the hydroxylamines can
reduce the mean infarct volume of a stroke when administered three hours
post stroke compared to controls.

Example 5

[0183]Inhibition of A.β Beta-Pleated Sheet Formation. The deposition
of amyloid β-peptide (Aβ) is associated with the development of
Alzheimer's disease. See, for example, G. G. Glenner et al. (1984)
Biochem. Biophys. Res. Commun., 120:885-890; and R. E. Tanzi (1989) Ann.
Med., 21:91-94. Accordingly, compounds which effectively disrupt the
formation of Aβ(1-40) or Aβ(1-42) beta-pleated sheets are
potentially useful for preventing and/or reversing such amyloid deposits.
Thioflavin T (ThT) is known to rapidly associate with beta-pleated
sheets, particularly the aggregated fibrils of synthetic Aβ(1-40).
This association gives rise to a new excitation maximum at 440 nm and to
enhanced emission at 490 nm. In this experiment, the ability of the
subject hydroxylamines to inhibit the association of ThT with synthetic
Aβ(1-40) or Aβ(1-42) is demonstrated by measuring changes in
fluorescence.

[0184]The experiments are performed using a CytoFluor II fluorescence
plate reader having the following parameters: Filters (Excitation and
Emission)=440 nm/20 and 490 nm/40; Gain=75; Cycle to Cycle Time=30 min;
Run Time=720 min (24 cycles) or dependent on exp. design; Plate=96 well.
Into each well is aliquoted 95 μL of ThT (3 μM) prepared in PBS (pH
6.0), 2 μL of the compound to be tested (10 μM) prepared with 0.05%
of methylcellulose in PBS (pH 6.0), and 3 μL of Aβ(1-40)(3 μg)
prepared with dH2O. The fluorescence measurement begins when the
Aβ(1-40) is added and continues for a total of 12 hours. The percent
inhibition of beta-pleated sheet formation is calculated from the
relative fluorescence unit difference between aggregation in the presence
and in the absence of the test compounds. The data show that compounds
prepared in Examples 1, 2 and 3 above inhibit Aβ(1-40) beta-pleated
sheet formation compared to the controls. In experiments conducted in a
similar manner using Aβ(1-42) instead of Aβ(140), the compounds
similarly inhibited Aβ(1-42) beta-pleated sheet formation compared
to the controls.

[0186]To the cells (7 DIV) is added 30 μM of Aβ(25-35) dissolved
in dH2O (stored at -20° C.) and 100 μM of a test compound
(e.g., a compound of Example 1, 2 and 3 above) in 1% methylcellulose.
Controls are also conducted without the test compound. The percentage of
morphologically viable neurons is determined counting the number of
viable neurons after 96 hours treatment compared to the number of neurons
before treatment in the same premarked culture regions (three
regions/culture, n=6). The data show that the hydroxylamines reduced
Aβ(25-35)-induced neuronal cell loss compared to the controls. In
experiments conducted in a similar manner using Aβ(1-40) instead of
Aβ(25-35), the compounds prepared in Example 1-3 above also reduce
Aβ(1-40)-induced neuronal cell loss compared to the controls.

Example 7

[0187]Reduction of Inflammation. In Alzheimer's disease, stroke and
multiple sclerosis, researchers have implicated an inflammatory response
in the etiology of the disease. See, for example, P. S. Aisen et al.,
(1994) Am. J. Psychiatry, 151:1105-1113; D. W. Dickson et al., (1993)
Glia, 7:75-83; and S. D. Yan et al., Proc. Natl. Acad. Sci. USA, 94, 5296
(1997). This response has been modeled in cell culture by utilizing
various factors to simulate the inflammatory response. Such factors
include lipopolysaccharide (LPS), an agent known to cause the expression
of nitric oxide and other cytokines; and interferon γ(INF-γ),
another agent implicated in the inflammatory/cytokine response. This
example demonstrates the ability of subject hydroxylamines to reduce the
inflammation caused by LPS and INF-γ.

[0188]In this experiment, the cell culture system is composed of E16 rat
pure cortical neuronal cells (treated with 10 μM Ara C to retard
astrocyte growth) that are plated on a confluent bed of two week old
cortical glial cells prepared from the cortices of 1 day old rat pups and
allowed to grow for one week. To these cells is added LPS (20 μg/mL),
IL-1β (40 mg/pg/mL), and INF-γ (200 U/mL), either with or
without 100 μM of the test hydroxylamine. Two days later, cell
viability was assessed using the lactate dehydrogenase (LDH) assay to
monitor cytosolic protein leakage due to cell membrane damage. The
results show that the hydroxylamines reduced the inflammation caused by
LPS and INF-γ compared to the control.

Example 8

[0189]Reduction of β-Amyloid-Induced Increased Cytokine Release. This
experiment demonstrates the ability of the hydroxylamines to reduce the
β-amyloid-induced increased release of cytokines, such as
interleukin-1β (IL-1). THP-1 cells, a human monocyte cell line from
American Type Culture Collection, are grown in RPMI-1640 medium plus 10%
fetal bovine serum (FBS, not heat-inactivated) in T-flasks. The medium is
changed every two days by spinning down (800 rpm, 5 minutes) the cells
and added the same fresh medium. Alternatively, the cultures are
maintained by the addition of fresh medium. The cultures are maintained
at a cell concentration ranging from between 1×105 and
1×10 cells/mL. Because sera may contain unknown factors which can
affect macrophage/monocyte IL-1 production, the FBS is reduced to 5% for
24 hours. The FBS is further reduced to 2% over two days prior to
starting each experiment. The cells are collected by centrifugation and
resuspended to 2% FBS. Cell numbers are calculated and cells plated on
24-well plates (3×105 cells/0.6 mL/well). Cells are then
treated with LPS (0.5 μg/ml or 0-10/g/ml for LPS dose-response
experiments) alone or in combination with Aβ peptides (54 μM or
0.05-5 μM for dose-response experiments). When determining the effect
of the hydroxylamines on cytokine release, 100 μM of the hydroxylamine
is added with the LPS and Aβ25-35 and this mixture incubated for 48
hours prior to performing ELISA.

[0190]IL-1β secretions into medium by LPS-stimulated THP-1 cells, in
the presence or absence of amyloid peptides and a test compound, are
assayed with a commercially available ELISA kit (R & D Systems). Briefly,
a microtiter plate coated with a murine monoclonal antibody to human
IL-1β is supplied by the manufacturer. Standards and samples are
pipetted into the wells and any IL-1β present bound by the
immobilized antibody. Unbound proteins are washed away and a horseradish
peroxidase-linked polyclonal antibody specific for IL-1β added to
the wells to "sandwich" the IL-1β bound in the initial step. After
washing to remove any unbound antibody-enzyme reagent, a substrate
solution (1:1 hydrogen peroxide--tetramethylbenzidine, v/v) is added to
the wells and color developed in proportion to the amount of IL-1β
bound in the initial step. Color development is stopped with 2N sulfuric
acid and the optical density of the standard and the test samples
measured at 450 nm. The amounts of IL-1β present in the samples are
calculated based upon a standard curve. Assays are run in quadruplicate
wells. The data show that the hydroxylamines reduce the
β-amyloid-induced increased release of interleukin-1β compared
to the controls.

Example 9

[0191]Reduction of Locomotor Impairment Due to Aβ-Peptide. This
experiment demonstrates the ability of the hydroxylamines to reduce the
in vivo impairment of animals treated with Aβ-peptide. Male
Sprague-Dawley rats (250-400 g) are given an ipsilateral injection of 20
μg of Aβ(25-35) into their substantia nigra. Prior to the
injection, the rats are fasted overnight and then each received an oral
treatment of the hydroxylamines (prepared in Examples 1, 2 and 3 above,
10-100 mg/kg) dissolved in aqueous 1% methyl cellulose or the vehicle
alone, one hour before and three hours post the Aβ-peptide
stereotaxic injection. One week after treatment, the rats are dosed s.c.
with 0.5 mg/kg apomorphine (dissolved in 0.1% vitamin C in isotonic
saline) and the circling reflex monitored using a Rotorat computerized
behavioral monitoring apparatus for the time period between 15 and 30
minutes of being placed in the arena. Impairment of the animals due to
Aβ-peptide is determined by measuring the number of rotations over
the 15 minute period. A higher number of rotations per period indicates
more physical impairment. The results show that the hydroxylamines
reduced the number of rotations per period and hence, the locomotor
impairment, of rats injected with Aβ(25-35) compared to
Aβ(25-35)-treated controls.

Example 10

[0192]Reduction of Spatial Learning Deficit. This experiment demonstrates
the ability of the hydroxylamines to reduce spatial learning deficiencies
in vivo. Treatment of rats with N-nitro-L-arginine, a nitric oxide
synthase inhibitor, is known to cause a deficit in spatial learning. See,
for example, G. A. Bohme et al., (1993) PNAS, 90:9191-9194. Rats treated
with N-nitro-L-arginine wander aimlessly throughout their enclosure
whereas untreated rats spend most of their time in the quadrant in which
they are initially placed and stay away from the open area in the middle
of the enclosure. This N-nitro-L-arginine-induced spatial learning
deficit is used as a model for learning deficits caused by Alzheimer's
disease and other dementias.

[0193]In this experiment, 10 mg/kg of a hydroxylamine or a control are
administered 30 min before each of nine doses of N-nitro-L-arginine (100
mg/kg. iip.). The results show that rats dosed with N-nitro-L-arginine
wander equally around the perimeter of the enclosure and readily cross
the center of the field. In contrast, rats treated with the
hydroxylamines show a preference for the area of the enclosure into which
they were first placed and rarely cross the center of the enclosure. This
behavior is essentially the same as rats treated with a saline control
(i.e., without N-nitro-L-arginine). These results demonstrate that the
hydroxylamines prevent the spatial learning deficit caused by
N-nitro-L-arginine.

Example 11

[0194]Prevention of MBP-Induced Experimental Allergic Encephalomyelitis.
Multiple sclerosis (MS) is a chronic inflammatory CNS disorder caused by
demyelination in the brain and spinal cord. The disease is characterized
by progressive CNS dysfunction, including muscular weakness, tremor,
incontinence, ocular disturbances, and mental dysfunction, with
remissions and exacerbations. At present, the only treatment for MS is
physical therapy.

[0195]Experimental allergic encephalomyelitis (EAE) induced by injection
of myelin basic protein (MBP) or MBP peptide fragments is reported to be
a useful model for MS. See, for example, D. E. McFarlin et al.,
"Recurrent Experimental Allergic Encephalomyelitis in the Lewis Rat," The
Journal of Immunology, 113(2): 712-715 (1974). This experiment
demonstrates the ability of the hydroxylamines to prevent MBP-induced
EAE.

[0196]Acclimated female Lewis rats, (Harlan; 200-250 g) are used in this
experiment since this strain of rat is genetically highly susceptible to
EAE. In the experiment, 100 mg/kg of the hydroxylamines (prepared in
Examples 1, 2 and 3 above) or a vehicle alone (control) is administered
to once a day from days 4 to 18. On day 1, the rats receive an injection
of 100 μg of MBP peptide, from guinea pig brain, plus 500 μg of
H37RA Mycobacterium in 0.10 ml complete Freund's adjuvant divided equally
between the two hind foot-pads.

[0197]The rats are evaluated on a 0-6 scale every day after day 7 until
day 18 (effects usually begin day 10 and peak day 15). See E. Heber-Katz,
"The Ups and Downs of EAE," International Reviews Immunology, 9: 277-285
(1992). These results show that the hydroxylamines completely
counteracted the effect of MBP in this test.

Example 12

[0198]Prevention of Weight Loss. Animals exposed to MBP or MBP peptide
exhibit significant weight loss as compared to controls exposed to
Freund's adjuvant alone. To determine if the hydroxylamines prevented
such weight loss, the animals in the EAE model described in Examples 1, 2
and 3 above were weighed daily. The results show that those animals
receiving the hydroxylamines exhibit normal or above normal weight gain
whereas the animals receiving MBP without the hydroxylamines showed
serious weight loss.

Example 13

[0199]Reduction of Learning Deficit in Autoimmune Mice. This experiment
demonstrates the ability of the hydroxylamines to reduce learning
deficiencies in autoimmune mice. Male MRL/MpJ controls and Fast mutation
mice were either dosed orally with 1% methylcellulose ("MC") or with 100
mg/kg of the hydroxylamines (prepared in Examples 1, 2 and 3 above, "test
compound") in 1% methylcellulose for 9-10 weeks. Following dosing,
animals of approximately 4 months of age are tested in an active
avoidance T-maze. In the one day test, animals are analyzed for
acquisition to avoid shock within the first five trials of the test. The
data reveal that animals administered the hydroxylamines show a 50%
protection in acquisition learning deficit compared to Fas mutated
animals receiving only 1% methylcellulose.

Example 14

[0200]Comparing in vivo the efficacy of subject hydroxylamines, PBN, and
two monosulfonate PBN compounds as agents for protecting against neuron
loss following brain ischemia and reperfusion injury. The test procedure
is that reported by W. Cao, J. M. Carney, A. Duchon, R. A. Floyd and M.
Chevion as "Oxygen free radical involvement in ischemia and reperfusion
injury to brain, Neuroscience Letters, 88 (1988), 233. In the experiments
a test compound is administered to groups of six gerbils i.p. as a single
dose 30 min before 5 min bilateral carotid occlusion. The density of
neuronal nuclei in a 100 micron is measured. Two controls are
present--controls which receive no test compound and controls which
receive no test compound and no brain ischemia. The compounds of the
invention show advantages as compared to the prior art compounds. These
results show a clear increase in potency for neural protection for the
subject hydroxylamines compared to PBN and two closely related analogs
and less in toxicity compared to PBN.

Example 15

[0201]Comparing the subject hydroxylamines to PBN and two sulfonate
analogs in post-ischemia treatment. The general method described above is
used but the test compounds are administered i.p. as a single dose 30 min
after reperfusion following 5 min ischemia. The results show that the
subject hydroxyamines again more potent at low doses and more potent and
less toxic at high doses.

Example 16

[0202]Comparing the subject hydroxylamines with PBN to determine the
relative effectiveness for protection of neuronal loss when administered
i.v. 60 min after reperfusion onset following 5 min ischemia in gerbils
using the general test method described above. The results illustrate
that the subject hydroxylamines are of significantly greater therapeutic
benefit in a clinical treatment setting following injury to the brain.
Neither PBN nor the subject hydroxylamines have an effect on neuronal
density in control gerbils without brain injury.

Example 17

[0203]Brain injury can manifest itself as behavioral changes. In this
experiment, young adult (3-4 months of age) gerbils are tested to
determine their ability to perform an 8-arm maze test 24 hours following
an ischemic event as described above As compared to nonischemic animals,
when untreated they committed many more errors. PBN and subject
hydroxyamines are administered to some of the test animals. Gerbils
treated with high doses of the hydroxylamines have error levels
indistinguishable from those of nonischemic animals. PBN is less
effective. This shows that subject hydroxylamines can protect against the
loss of temporal/spatial short term memory following ischemia (24 hours
post) errors in 8-arm radial maze test of young gerbils following 5 min
ischemia.

Example 18

[0204]The ability of the subject hydroxylamines to reduce infarct volume
following an ischemic event. While PBN and the hydroxylamines are both
effective at low doses, at high doses hydroxylamines gave the best
protection and PBN was toxic.

Example 19

[0205]In this study, subject hydroxylamines and PBN are compared for their
ability to impart lethality protection (% survived) in aged gerbils
(18-24 months of age, n=12/group) from 10 min ischemia when given 30 min
before ischemia. The hydroxylamines are superior at all dose levels and
achieve complete protection at high levels while PBN is only partially
effective.

Example 20

[0206]An important advantage of the subject hydroxylamines as compared to
PBN, is its markedly diminished toxicity. Acute lethality in C57BL/6 L
mice is determined based upon varying sizes of single i.p. doses of
hydroxylamine. PBN shows significant toxicity at 560 mg/kg dose levels.
The hydroxylamines show no toxicity at doses nearly twenty times as
great.

Example 21

[0207]Another undesirable systemic effect which has been observed in vivo
with nitrone radical traps is a depression in body temperature. This
toxicity can have serious health consequences and also can complicate
diagnosis of other conditions. The subject hydroxylamines are
administered to mice and gerbils at levels as high as 1000 mg/kg with no
measurable temperature decrease. In contrast, PBN gives up to an
8° C. decrease in body temperature at a does of only 500 mg/kg.

Example 22

[0208]Hydroxylamines effectiveness in the treatment of conditions
characterized by protracted low grade oxidative stress upon the central
nervous system and gradual progressive central nervous system function
loss by effectiveness in a model for Alzheimer's disease ("AD"). Studies
have demonstrated that there is an age-associated increase in protein
oxidation and loss of enzyme activities in the brain of aged individuals.
Tissue cultures of fibroblasts from aged individuals and red blood cells
of different ages both show an exponential increase in protein carbonyl
content (a measure of protein oxidation) and a decrease in marker enzyme
activities. Brain protein oxidation progressively increases over the life
span of the individual. The role of abnormal amyloid precursor peptide
processing and metabolism in AD has also been explored in a number of
different models. In vitro studies using embryonic hippocampal neuronal
and neuronal/glial cultures have demonstrated that βAP 1-40 produces
cytotoxicity over an extended period of co-incubation. When this peptide
is infused into rat brains, lesions are produced. Some of the proposed
breakdown fragments of PAP are also neurotoxic, e.g. PAP (25-35). The
neurotoxicity appears to be both mediated via glutamate receptors, and
also by non-glutamate receptors mechanisms. Confocal microscopy studies
of neuronal cultures have demonstrated that exposure to βAP (1-40)
results in oxidative stress.

[0209]It has been demonstrated that βAP fragments can directly
inactivate glutamine synthetase (GS) and creatine kinase (CK) in tissue
extracts and in cultured hippocampal neurons and glia. While the
hydroxylamines and PBN each show the ability to protect GS and CK against
the effects of βAP fragments, the hydroxyamines give complete
protection and in fact can at least partly reverse the effects of
oxidation. In contrast, PBN's effectiveness is quite limited as it
asymptotically levels out at a substantially incomplete level of
protection.

Example 23

[0210]The effectiveness of the subject hydroxylamines in preventing
central nervous system damage caused by stroke. Rat focal ischemia
results show the efficacy of subject hydroxylamines in a rat focal
ischemia model. In this model, Sprague Dawley rats (200-300 g) undergo a
permanent middle cerebral artery occlusion (MCAO) to induce a focal
stroke. Subject hydroxylamines are administered after the permanent
occlusion as first an intraperitoneal (i.p.) bolus dose and then by
intravenous (i.v.) continuous infusion during the remaining time up to 24
hours post stroke. The doses used were either 100 mg/kg, i.p., followed
by 4.2 mg/kg/hr, i.v., or 10 mg/kg, i.p., followed by 0.42 mg/kg/hr, i.v.
The rats are sacrificed 3 days post stroke, the tissue processed
histologically using triphenyltetrazolium staining techniques, and the
infarct volume, the area of total cell necrosis, quantitated using image
analysis. The results of these experiments demonstrate that subject
hydroxylamines provide significant protection, approximately 70%.

Example 24

[0211]Evaluating the ability of the subject hydroxylamines to ameliorate
oxidation-caused side effects of anticancer therapy. Adriamycin is a
widely used anticancer agent. It is known to be very effective but it is
also known to have serious side effects arising from its tendency to
cause oxidative damage. These side effects include causing serious levels
of cardiac damage at high dose levels. These side effects have often
limited the use of this agent or limited the dose levels that can be
employed to levels which are below those desired for maximum
antineoplastic disease effectiveness.

[0212]Experiments demonstrate that the subject hydroxylamines are
effective at reducing the side effects of anticancer agents such as
adriamycin and permitting higher dose levels of adriamycin to be
tolerated by animals. C57BL/6J and DBA/2J male mice (35-40 g) are tested
for the acute lethal effects of adriamycin and the prevention of acute
lethality by pretreatment doses of subject hydroxylamines. Mice are
injected i.p with either saline or subject hydroxylamines 30 minutes
prior to administration of adriamycin. The acute lethality of adriamycin
ranges from 10 to 30 mg/kg. The LD50 for adriamycin in these tests
was found to be 25 mg/kg in both mouse strains. Hydroxylamines doses up
to 300 mg/kg, without adriamycin treatment, have no effect on survival in
the two mouse strains. Pretreatment with 30 and 100 mg/kg of
hydroxylamines produces dose related shifts in the adriamycin lethality
dose effect curve. A dose of 100 mg/kg of subject hydroxylamines produces
a 5-fold shift to the right (in the direction of reduced lethality).
Thus, the combination of subject hydroxylamines with adriamycin results
in a marked increase in the maximally tolerated dose. These higher doses
are in the range that would effectively kill multi-drug resistant tumors.

[0213]Comparative Tests. PBN pretreatment results in a slight shift to the
right in the adriamycin does-effect curve. While the subject
hydroxylamine dosages can be increased to 300 mg/kg in combination with
adriamycin, there is an upper limit for this combination with PBN. A dose
of PBN of 100 mg/kg produces slight sedation and 300 mg/kg yielded marked
sedation and some combined toxicity (10-20% lethality).
Hydroxylamines/adriamycin does not produce any combined toxicity at doses
of hydroxylamine of up to 300 mg/kg.

Example 25

[0214]Safety Testing. The subject hydroxylamines and PBN are tested for
their acute toxicity in male Sprague Dawley (200-300 g) rats. The
compounds are administered at 1000 mg/kg, i.p., to groups of 6 rats.
After 3 days lethality is assessed. Hydroxylamines causes no lethality,
while PBN is lethal to 5 of the 6 rats used in this test. These data
confirm the gerbil data in that the hydroxylamines have higher safety
than PBN.

Example 26

[0215]Delaying Senescence: Detailed Experimental Protocols. This example
shows that N-t-butyl hydroxylamine, a hydrolysis product of
α-phenyl-N-t-butyl nitrone, delays senescence in IMR90 human lung
fibroblasts. The ability of N-t-butyl hydroxylamine to exert this effect
at concentrations much lower than that used for PBN together with
increased potency of PBN preparations with longer storage time suggests
that this decomposition product mediates PBN's purported actions on IMR90
cells. Benzaldehyde was without effect and in high concentrations was
toxic to the cells. Related N-hydroxylamines, N-benzyl hydroxylamine and
N-methyl hydroxylamine, have also been found to be active.

[0217]Cultivating IMR90 cells in culture--Normal human epithelial
fibroblasts (IMR90) cells were obtained from the Coriell Institute for
Medical Research at a population doubling level (PDL) of 10.85. The PDLs
were calculated as log2(D/Do), where D and Do are defined
as the density of cells at the time of harvesting and seeding,
respectively. Stock cultures were grown in 100 mm Corning tissue culture
dishes containing 10 ml of Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% (V/V) fetal bovine serum (Hyclone). Stock cultures
were split once a week when near confluence. Cells were harvested by
trypsinization for 5 min at 37° C., immediately collected in 5 ml
complete DMEM, washed once with 5 ml complete DMEM and incubated for
10-15 min at 37° C. to allow the cells to recover.

[0218]To test the effect of hydroxylamines on replicative life span, IMR90
cells were seeded at 0.5×106 per 100 mm dish. N-hydroxylamines
(N-t-butyl hydroxylamine, N-benzyl hydroxylamine, N-methyl
hydroxyl-amine) were added either individually (final concentration 10 or
100 μM) or in a combination of all three N-hydroxylamines (30 μM
each). The cultures were split after 7 days and seeded with fresh medium
supplemented with the hydroxylamines described above. In other
experiments the medium of the cultures was replaced after 3 days of
seeding with fresh medium and with fresh N-hydroxylamines, the split was
done as usual after 7 days from seeding. The effect of PBN on life span
was tested as in (5).

[0219]To determine the effect of H2O2 on the replicative life
span, cells were first seeded with fresh medium with or without
N-hydroxylamines (see above) for a week. Next, cells grown with or
without N-hydroxylamines were each split into two additional groups and
then either; 1) treated with 20 or 30 μM H2O2 or 2) without
treatment with H2O2.

[0220]Analysis of Aconitase activity in tissue culture treated with
N-hydroxylamine-Aconitase was measured as described by Gardner et al.
(25). Briefly; IMR90 cells were grown with or without N-hydroxylamine as
described above. After 12 weeks of treatment the cells were washed twice
by cold PBS and scraped from the dishes by cell scraper. The cells
(3-4×106) were collected by centrifugation and resuspended
into 200 μl of ice cold 50 mM tris, pH 7.4/0.6 mM MnCl2/20 μM
fluorocitrate supplemented with antiprotease mixture (leupeptin,
pepstatin and PMSF, 1 μg each). The cells were disrupted by three
cycles of sonication for 3-5 sec at low output separated by 1 minute of
incubation in ice. Then the lysate was spun at 12000 g for 5 min in
4° C. and the supernatant was used to measure total soluble
protein and aconitase activity. In general 60-100 μg of protein are
adequate to readily detect aconitase activity as described (25).

[0221]Analysis of the age-dependent changes in the steady state level of
oxidants and mitochondrial membrane potential in IMR90 cells by
FACS--IMR90 cells were trypsinized and resuspended into complete DMEM.
For each condition, two tubes were prepared with 1×106 cells
each. Tubes were then spun at 250 g for 10 min at room temperature and
supernatant was replaced with 1 ml of Hanks Balanced Salt Solution (HBSS)
without Ca++ or Mg++. Rho123 (20 μl of 525 μM stock; 10.5
μM final concentration) was added to one tube and DCFH (20 μl of
1.25 mM stock; 25 μM final concentration) was added to the other tube.
The cells were then incubated in the dark in a water bath at 37°
C. for 30 min followed by cell resuspension and centrifugation at 250 g
for 10 min at room temperature. The supernatant (500 μl) was removed
from each tube and the cells were resuspended in the remaining 500 μl
before FACS analysis on a FACSort analyzer (Becton Dickinson, San Jose,
Calif.). Cell Quest was used for data acquisition and analysis. The data
is reported as the mean of the channel of the fluorescence histogram
obtained. Fluorescence output was calibrated with LinearFlow Green Flow
cytometry Intensity Calibration Particles (Molecular Probes, Eugene,
Oreg.).

[0222]Measurement of apurinic/apyrimidinic (AP) sites in IMR90
cells--Briefly, Aβ sites were measured as follows: IMR90 cells
(1-2×106) in 0.5 ml PBS/5 mM glucose were incubated with 3 mM
Aldehyde Reactive Probe (ARP) for 60 min at 37° C. The cells were
then collected by centrifugation at room temperature and washed twice
with 1 ml PBS. DNA was isolated by the QIAamp blood kit as suggested by
the manufacturer. DNA was quantified by Picogreen and 1 μg was
transferred into 200 μl of elution buffer (10 mM Tris, pH 8.9), mixed
with 14 μl of 5 M NaCl (the mole ratio of NaCl/dNTP should be
25000-30000) and incubated for 60 min at room temperature with 30 μl
of freshly prepared avidin-HRP (ABC kit), prepared as described by the
manufacturer but with avidin-HRP concentrations diluted 1:3 and the
incubation volumes scaled down to 1 ml. The DNA-avidin-HRP complex
(DNA-HRP) was separated from unbound avidin-HRP by gently mixing 65 μl
of 1 mM DAPER(N,N'-bis(3,3'-(dimethylamino)-propylamine)-3,4,9,10-perylen-
e-tetra carboxylic diimide) with the DNA and incubated at room temperature
for 5 min. The DNA-DAPER precipitate was then collected by centrifugation
for 5 min at 12,500 g, and washed twice with 1.5 ml of 0.17 M NaCl/20 mM
Tris/0.25% Tween-20/1% BSA, pH 8. The precipitate of DNA-HRP was
suspended in 500 μl of ice cold 50 mM Na--Citrate, pH 5.3 and
sonicated at output 1-2 watts for 5 sec (Sonifier cell Disrupter, model
w185D, Branson) and cooled immediately. HRP activity was measured as an
indicator of AP sites in DNA-HRP by using the chromogenic ImmunoPure TMB
or the fluorogenic QuantaBlu Substrate kits. The background control was
established by performing a parallel analysis on calf thymus DNA. The
standard curve for AP sites was constructed with 100 ng of DNA standard
containing a known amount of uracil suspended in 50 μl of 10 mM
Na2HPO4, pH 7.5. The standard DNA was incubated with 25 μM
spermine for 3 min and then with 3 U of uracil-DNA N-glycosylase (UNG)
for 20 min at 37° C. to catalyze the removal of uracil residues
and generate AP sites. The resulting "AP enriched" DNA was incubated with
3 mM ARP for 45 min at 37° C. The standard DNA-ARP adducts were
isolated from unbound ARP by QIAamp columns (without the protease step)
and quantified. The number of ARP sites were corrected for the loss of
DNA during isolation (10-20% loss). The biotinylated DNA was incubated
with avidin-HRP and processed as above.

[0223]Reduction of cyt cIII by superoxide radical--Superoxide radical
was generated by the reaction of xanthine (120 μM) with xanthine
oxidase (XO, 0.06 U). The reaction was performed at 25° C. in a
final volume of 1 ml PBS containing 40 μM cIII. The reaction was
started by the addition of the substrate xanthine (X). N-hydroxylamines
were added just before the addition of X. The initial rate of reduction
of cyt cIII was determined based on the linear change in absorbance
at 550 nm.

[0224]In order to test the effect of N-hydroxylamines on the spontaneous
oxidation of cytochrome c, a complete reduction of cyt cIII was
achieved by incubating the X/XO system for 4-5 min at 25° C.
Auto-oxidation of cyt cII is associated with a decrease in
absorbance at 550 nm. Reduced cytochrome c was incubated at 25° C.
with or without 2 or 3 mM N-hydroxylamines and the auto-oxidation was
followed by spectrophotometer. The rate of reduction of cytochrome c by
different concentrations of each N-hydroxylamine was measured by the
increase in absorbance at 550 nm.

[0226]N-t-butyl hydroxylamines, and other N-hydroxylamines delay
senescence of IMR90 cells--N-t-butyl hydroxylamine, N-benzyl
hydroxylamine and N-methyl hydroxylamine (N-hydroxylamines, scheme 1) at
100 μM (added once per 7 days) delay senescence of IMR90 cells by at
least 17-20 PDLs. The concentration of PBN required to achieve nearly a
similar gain in PDLs is 8 times higher than N-hydroxylamines (table 1 and
(5)).

TABLE-US-00015
TABLE 1
The gain in PDLs of cultured IMR90 cells after continuous
cultivation with PBN and N-t-butyl hydroxylamines compared
to control untreated cells. IMR90 cells were cultured in
the presence of various compounds and PDL followed until
senescence. PDL were calculated as described in methods.
Data from a representative experiment is shown.
Treatment Gain in PDLs
800 μM PBN 14.8
200 μM PBN 2.4
100 μM NtBHA 19.7
10 μM NtBHA 5.8
NtBHA = N-t-butyl hydroxylamine.

The minimal concentration of N-hydroxylamines required to achieve a gain
of 5-7 PDLs above the untreated control was 20 times lower than that for
PBN (200 μM) to achieve 2-3 PDLs (table 1 and (5)). For each of the
three N-hydroxylamines when IMR90 cells were treated at 25 μM every 3
days, it was twice as efficient as 100 μM every 7 days. None of the
N-hydroxylamines tested were toxic at the concentration applied to the
cells, as measured by PDL; whereas benzaldehyde, the co-product of PBN
hydrolysis, was without effect or toxic at high concentrations. All the
N-hydroxylamines, at the concentrations tested, were more effective than
PBN in delaying senescence. The N-hydroxylamines studied appear to be
equally efficient in delaying senescence, with a variation only when
cells are close to senescence (late PDLs).

[0227]A simultaneous treatment of the cells with all three of the
N-hydroxylamines (30 μM each) yielded results similar to single
treatments, delaying senescence by 14-17 PDLs. In contrast, at
concentrations equivalent to the N-hydroxylamines, the isomeric
O-hydroxylamines (O-t-butyl hydroxylamine, O-benzyl hydroxylamine and
O-methyl hydroxylamine) were found to accelerate senescence.

[0228]N-t-butyl hydroxylamines, and other N-hydroxylamines delay
senescence-dependent change in mitochondria-Senescence-dependent change
in mitochondria of IMR90 cells was estimated by Rho123. The Rho123
fluorescence that accumulated in the cells was measured weekly by FACS
for a total of at least 8 weeks and plotted against the current PDL
(i.e., age of the cells). The age-dependent incorporation of Rho123 in
IMR90 is biphasic. This is characterized by a slow and linear increase at
early PDLs, followed by a shorter and steeper phase at late PDLs. Linear
regression analysis was used to calculate the rate of Rho123 accumulation
as a function of PDL (Table 2).

The regression analysis was based on early PDLs only, late PDLs were not
included in the analysis. The increment in accumulation of Rho123
indicates a senescence-dependent change in the mitochondria of IMR90
cells as they become senescent. N-hydroxylamines delay these changes in
mitochondria, and the rate of Rho123 accumulation as a function of
senescence decreased by 70%, 69%, and 52% for N-t-butyl hydroxylamine,
N-benzyl hydroxylamine and N-methyl hydroxylamine, respectively (Table
2).

[0229]N-t-butyl hydroxylamines, and other N-hydroxylamines decrease
formation of oxidants and oxidative DNA damage in IMR90 cells--The level
of oxidants was measured each week by estimating the oxidation of DCFH
(27) in the living cells. Measurements of fluorescence of oxidized DCFH
were made weekly by FACS for total of at least 8 weeks and plotted
against the current PDL (i.e., age of the cells), and a biphasic curve,
similar to that seen with Rho123 fluorescence, was observed with DCFH
fluorescence. A linear regression analysis was used to calculate the
initial linear rate of DCFH oxidation as a function of PDL (Table 2). The
regression analysis was based on early PDLs only. Late PDLs were not
included in the analysis. IMR90 cells treated continuously with
N-hydroxylamines exhibit a slower rate of formation of oxidants compared
to control cells. The percent of decrease in the rate of oxidants
formation from control are 88%, 95% and 79% for N-t-butyl hydroxylamine,
N-benzyl hydroxylamine and N-methyl hydroxylamine, respectively (Table
2). The level of AP sites in DNA can be used as a measure of the level of
oxidative damage. IMR90 cells treated simultaneously with the three
N-hydroxylamines (30 μM each) showed a 52% reduction in AP sites
compared to PDL matched control cells.

[0230]N-t-butyl hydroxylamines and the other N-Hydroxylamines increase the
activity of aconitase in IMR90 cells-A 2-3 fold age-dependent decline in
the activity of aconitase is seen in old (high PDL) compared to young
(low PDL) IMR90 cells. The age-dependent decline in the activity of
aconitase to a large extent was prevented when the cells were grown with
N-hydroxylamines. The efficiency of protecting aconitase from inhibition
was as follows; N-t-butyl hydroxylamine (90%) 2 N-benzyl hydroxylamines
(75%)>>N-methyl hydroxylamine (17%). This order is comparable to
the relative efficiencies for the ability to delay senescence.

[0232]The GSH/GSSG ratio increased because of a decrease in the level of
GSSG in treated cells compared to untreated cells. No change in the level
of GSH was observed between the treated and control groups (Table 3).
When the cells were treated simultaneously with the three
N-hydroxylamines a similar effect on GSH metabolism was observed.

[0233]IMR90 cells treated with N-t-butyl hydroxylamine and N-benzyl
hydroxylamine are resistant to hydrogen peroxide-Hydrogen peroxide,
applied at low concentrations (20 μM or 30 μM in fresh medium) once
a week to control IMR90 cells accelerated senescence. The
H2O2-induced senescence was attenuated when these cells were
continuously treated with N-t-butyl hydroxylamine, N-benzyl hydroxylamine
or both compounds+N-methyl hydroxylamine.

[0234]N-Hydroxylamines inhibit reduction of cyt cIII by superoxide
radical--N-hydroxylamines at relatively high concentrations (5-10 mM)
were able to inhibit the reduction of cyt cIII by X/XO, a system
that generates superoxide radical. The catalytic activity of the enzyme
XO was not inhibited by N-hydroxylamines, as judged from the rate of
formation of uric acid (the co-product with O2-) in the
presence or absence of N-hydroxylamines (data not shown). Moreover,
N-hydroxylamines prevented auto-oxidation of cyt cII.
N-hydroxylamines were able to reduce cyt cIII directly to cyt
cII, which explains their ability to delay the oxidation of reduced
cyt cII. The reduction of cytochrome c by N-hydroxylamines was
equally efficient under aerobic and anaerobic condition or in the
presence of iron chelator (DTPA). A differential ability to reduce
cytochrome c was observed for the three different N-hydroxylamines,
N-t-butyl hydroxylamine being somewhat less efficient.

[0235]In this example, we have demonstrated that the products of PBN or
PBN/-OH hydrolysis, N-t-butyl hydroxylamine, but not benzaldehyde,
delays the replicative senescence of human lung fibroblasts at
concentrations 20 times lower than PBN. Thus N-t-butyl hydroxylamine is
much more effective than PBN in delaying senescence of IMR90 cells and
appears to be the active component in old preparations of PBN. Other
N-hydroxylamines tested (not related to PBN, e.g., N-benzyl hydroxylamine
and N-methyl hydroxylamine), were also able to delay the senescence of
IMR90 cells. Thus, we conclude that the N-hydroxylamine functional group
is responsible for their biological activity. On the other hand, although
PBN is a spin trap and an antioxidant, none of the well known spin traps
or antioxidants studied (ascorbic acid, vitamin E, catalase,
2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) and
4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-OH-TEMPO) can delay
senescence of IMR90 cells as does PBN. These results indicate that the
effect of PBN on IMR90 cells is due to N-t-butyl hydroxylamine and not
PBN itself.

[0236]In order to gain more insight into the effect of N-hydroxylamines on
cells, we assessed the status of different cellular parameters in cells
that have continuously been grown with medium supplemented with
N-hydroxylamines compared to controls. We show that, concomitantly with
delayed senescence by N-hydroxylamines, the PDL-dependent formation of
oxidants was decreased as estimated by DCFH oxidation (Table 2), and an
increase in the GSH/GSSG ratio (Table 3). The age-dependent decay in
mitochondria was delayed as estimated by Rho123 accumulation (Table 2)
and by the inhibition of the age-dependent decline in the activity of
aconitase. The level of AP sites in DNA of cells treated with
N-hydroxylamines was also 52% lower than that of the control cells. The
increase in the ratio GSH/GSSG by treatment with N-hydroxylamines was due
to a decrease in the steady-state level of GSSG, without changing the
concentration of GSH. In addition N-hydroxylamines prevented the
age-dependent decline in aconitase activity in IMR90. Aconitase is an
enzyme essential for the Krebs cycle and highly abundant in mitochondria
compared to cytosol (28). Its iron-sulfur cluster is known to be damaged
by superoxide radical and ONOO- (25,29,30). The mitochondrial enzyme
is more sensitive to inhibition by superoxide radical and oxidative
modification compared to the cytosolic enzyme (29,31). These findings
indicate that N-hydroxylamines lower the endogenous level of oxidants in
mitochondria, thus protecting aconitase and causing less GSH to be
oxidized to GSSG. Since aconitase plays an important role in the Krebs
cycle, changes in its activity will have a large impact on mitochondrial
and cellular metabolic pathways. N-hydroxylamines also protect IMR90
cells from H2O2-induced senescence, by acting as mitochondrial
anti-oxidants. This is further supported by the 79-95% decrease in the
rate of DCFH oxidation in N-hydroxylamine treated cells compared to
controls.

[0237]PBN has been shown to protect against oxidative damage in different
biological models as well at higher concentrations (5,32-34).
Interestingly, PBN inhibits formation of hydrogen peroxide at the level
of complex I in mitochondrial preparations which suggests a direct
interaction with mitochondria in vivo (10). The antioxidative effect of
N-t-butyl hydroxylamine can be attributed to a similar, though more
efficient, inhibition of superoxide formation by mitochondria in vivo,
resulting in less hydrogen peroxide being formed. We studied further the
interaction of N-t-butyl hydroxylamine (as representative of primary
N-hydroxylamines) with mitochondria in IMR90 cells. Intracellular
N-t-butyl hydroxylamine is maintained in the reduced form by
mitochondrial NADH and complex I. Since N-t-butyl hydroxylamine is stable
to auto-oxidation in a cell free system, this indicates that N-t-butyl
hydroxylamine cycles inside the cells between the oxidized and reduced
form. Complex I is a mitochondrial site that is implicated in the
formation of superoxide radical, indicating that N-t-butyl hydroxylamine
interacts with this site to prevent formation of superoxide radical, as
with the interaction of PBN with complex I (10).

[0238]The age-related increase in oxidative damage to mitochondrial DNA,
proteins and lipids is thought to be a major factor in organismal aging
(6,35-38). Since mitochondria are assumed to play a major role in the
formation of superoxide radical and suggested to contribute to aging, we
compared the senescence-dependent changes in mitochondria in control and
N-hydroxylamine treated cells. A PDL-dependent accumulation of Rho123 is
observed in IMR90 cells, which reflects a senescence-dependent change in
mitochondria (Table 2). This change may be due to age-dependent
mitochondrial swelling, or changes in the mitochondrial inner membrane
that elevates the non-specific binding of Rho123 to this membrane (6,39).
Accumulation of Rho123 was also observed in one fraction of isolated
hepatocytes from livers of old rats over hepatocytes from young rats (6).
When IMR90 cells were grown in medium supplemented with N-hydroxylamine,
a 52-70% slower rate of the age-dependent accumulation of Rho123 was
observed when compared to control cells. This indicates, in conjunction
with the protective effect on aconitase, that N-hydroxylamines interact
with mitochondria and delay the senescence-dependent changes to
mitochondria. Since mitochondria are a major source for free radical
formation, improving the mitochondrial status provides a significant
decrease in the level of oxidants in the cells (Table 2).

[0239]We also found that cyt cIII is reduced directly by
N-hydroxylamines independently of oxygen or iron, indicating that
superoxide radical is not an intermediate in the process. Reduction of
cyt cIII by N-hydroxylamines indicates that N-hydroxylamines can
interact in vivo with cytochrome c in addition to mitochondrial NADH.
Cyclic-N-hydroxylamines/cyclic-nitroxides are recycled by mitochondrial
ubiquinol and cytochrome oxidase (22,23), a mechanism of regeneration
that may be shared by the primary N-hydroxylamines used in the present
study. Our primary data show that mitochondrial NADH is involved in
keeping the intracellular N-hydroxylamines in reduced form.

[0240]N-hydroxylamines (5-10 mM) inhibit the reduction of cyt cIII by
superoxide radical, which was generated with xanthine/xanthine oxidase.
N-hydroxylamines do not inhibit the catalytic activity of xanthine
oxidase since the formation of uric acid (obligatory product with
superoxide radical) was not inhibited. This indicates that in vivo,
primary N-hydroxylamines (or their corresponding nitroxides), react with
superoxide radical, as is known for the
cyclic-hydroxylamines/cyclic-nitroxides. We find that N-t-butyl
hydroxylamine rapidly enters the cells and is concentrated by
approximately 5-fold. In order to test the contribution of superoxide
scavenging to the mechanism of senescence delay we tested two
cyclic-nitroxides as typical non-metal SOD mimics. Both of the
cyclic-nitroxides tested (TEMPO and 4-OH-TEMPO) did not delay the
replicative senescence of the cells (at 25 μM) and at high
concentrations (100 μM) were even toxic. This indicates that SOD mimic
activity (which gives H2O2) by itself can not account for the
protective effects and senescence delay observed with the primary
N-hydroxylamines used in this study. Consequently we conclude that
mitochondria are a primary target for N-hydroxylamines due to their
ability to slow the senescence-dependent changes to mitochondria, lower
oxidants and delay senescence of IMR90 cells.

[0241]Nitric oxide was proposed as a product of PBN decomposition and thus
was suggested to possess a role in the activity of PBN in vivo. N-t-butyl
hydroxylamine has also been shown to be oxidized by UV photolysis to
produce nitroso-tert-butane (tNB), which further decomposes to give
nitric oxide (11,13). The in vivo evidence for the formation of N-t-butyl
hydroxylamine-dependent (or PBN-dependent) nitric oxide has not been
demonstrated, and the evidence is circumstantial or based on in vitro
experiments (40,41). In order to assess if tNB contributes to the effect
of N-t-butyl hydroxylamine on IMR90 cells, the cells were grown in a
medium supplemented with tNB. We found that tNB is toxic at 50 μM, and
has no effect on the cells at much lower concentrations (10 μM). Thus,
tNB plays a negligible role in the mechanism underlying the biological
effect of N-t-butyl hydroxylamine.

[0242]The N-hydroxylamines used in this study all exhibit the ability to
delay cellular senescence. Cyclic-N-hydroxylamines (R2NOH) and their
respective nitroxides enhance the clinical recovery of damaged brains in
closed-head injury (42) and protect against oxidative damage induced by
H2O2 (43) but did not delay cellular senescence. This
emphasizes the remarkable feature of the primary N-hydroxylamines as
antioxidants. Harman in 1961 (44) reported that HNHOH (hydroxylamine)
possesses anticancer activity and delayed senescence in mice. On the
other hand O-hydroxylamines which possess a different functional group
(R--O--NH2), but the same alkyl groups (and benzyl group) as
N-hydroxylamines, do not affect the rate of senescence, the level of
oxidants or the changes in mitochondria in IMR90 cells. This further
indicates that the N-hydroxylamine functional group (R--NHOH) is involved
in the effect of delaying senescence in IMR90 cells. The alkyl and
aromatic groups of the primary N-hydroxylamines can affect their
oxidation-reduction potential, as is the case with cyclic
nitroxides/cyclic hydroxylamines (22). This ratio is also determined by
the oxygen status of the cell (24,45). In addition, the alkyl groups and
their different hydrophobicities can influence the intracellular location
of the N-hydroxylamines.

[0243]In summary, the anti-senescence effect of PBN on IMR90 cells can be
mimicked efficiently by N-t-butyl hydroxylamine, and other
N-hydroxylamines which indicates that the functional compound in the PBN
preparation is the N-hydroxylamine rather than PBN itself. Other
N-hydroxylamines were also effective in delaying senescence and
protecting IMR90 cells. The use of N-hydroxylamine also avoids the
benzaldehyde formed when PBN decomposes (41). The low doses of
N-hydroxylamine required make them desirable compounds for delaying aging
and protecting from oxidative damage. This is the first time that an
anti-aging activity has been attributed to a group of chemicals that
share a common functional group.

[0289]All publications and patent applications cited in this specification
and all references cited therein are herein incorporated by reference as
if each individual publication or patent application or reference were
specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it
will be readily apparent to those of ordinary skill in the art in light
of the teachings of this invention that certain changes and modifications
may be made thereto without departing from the spirit or scope of the
appended claims.